Cysteine String Protein Research Articles

Investigating the Expression Profiles of Cysteine String Proteins (CSPs) in Cochlear Tissue

ObjectiveThis study aims to explore the expression patterns of cysteine string protein alpha (CSPα) and cysteine string protein beta (CSPβ) in the mammalian inner ear, with an emphasis on their temporal dynamics during the developmental stages of C57BL/6 mice. MethodsWe utilized immunofluorescence staining to assess the localization and distribution of CSPα and CSPβ within the inner ears of C57BL/6 mice and miniature pigs. Additionally, this method facilitated the investigation of their temporal expression profiles. ResultsIn adult C57BL/6 mice and miniature pigs, CSPα and CSPβ were identified in the cytoplasm of inner hair cells and spiral ganglion cells, yet were absent in outer hair cells. Both proteins were found to colocalize with Ctbp2 on the basal side of the cytoplasm in inner hair cells' basilar membrane. Expression of CSPα was observed at the nerve fiber termini at the basilar membrane's base of inner and outer hair cells 10 days postnatally in C57BL/6 mice. Notably, expression of both CSPα and CSPβ in the cytoplasm of inner hair cells emerged on the 12th day post-birth, aligning with the timeline for registering cochlear potentials. The expression levels of both proteins increased with age, but were consistently absent in outer hair cells. Contrastingly, expression of CSPα and CSPβ was present in the cytoplasm of inner hair cells in miniature pigs as early as one day post-birth, yet remained absent in the three rows of outer hair cells. ConclusionCSPα and CSPβ exhibit predominant and specific expression in inner hair cells and spiral ganglion cells. A unique expression pattern was observed for CSPα, which was also present at the nerve fiber endings of both inner and outer hair cells. The developmental expression trajectory of CSPα and CSPβ in mouse inner hair cells is characterized by an initial absence, followed by a gradual increase. Moreover, the timing of expression onset between mice and miniature pigs indicates distinct temporal dynamics, suggesting a potential role in auditory development.

Decoding transcriptomic signatures of cysteine string protein alpha–mediated synapse maintenance

Synapse maintenance is essential for generating functional circuitry, and decrement in this process is a hallmark of neurodegenerative disease. Yet, little is known about synapse maintenance in vivo. Cysteine string protein α (CSPα), encoded by the Dnajc5 gene, is a synaptic vesicle chaperone that is necessary for synapse maintenance and linked to neurodegeneration. To investigate the transcriptional changes associated with synapse maintenance, we performed single-nucleus transcriptomics on the cortex of young CSPα knockout (KO) mice and littermate controls. Through differential expression and gene ontology analysis, we observed that both neurons and glial cells exhibit unique signatures in the CSPα KO brain. Significantly, all neuronal classes in CSPα KO brains show strong signatures of repression in synaptic pathways, while up-regulating autophagy-related genes. Through visualization of synapses and autophagosomes by electron microscopy, we confirmed these alterations especially in inhibitory synapses. Glial responses varied by cell type, with microglia exhibiting activation. By imputing cell-cell interactions, we found that neuron-glia interactions were specifically increased in CSPα KO mice. This was mediated by synaptogenic adhesion molecules, with the classical Neurexin1-Neuroligin 1 pair being the most prominent, suggesting that communication of glial cells with neurons is strengthened in CSPα KO mice to preserve synapse maintenance. Together, this study provides a rich dataset of transcriptional changes in the CSPα KO cortex and reveals insights into synapse maintenance and neurodegeneration.

Tissue distribution of cysteine string protein/DNAJC5 in C. elegans analysed by CRISPR/Cas9-mediated tagging of endogenous DNJ-14.

Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 family of molecular chaperones. CSP is enriched in neurons, where it mainly localises to synaptic vesicles. Mutations in CSP-encoding genes in flies, worms, mice and humans result in neuronal dysfunction, neurodegeneration and reduced lifespan. Most attention has therefore focused on CSP's neuronal functions, although CSP is also expressed in non-neuronal cells. Here, we used genome editing to fluorescently tag the Caenorhabditis elegans CSP orthologue, dnj-14, to identify which tissues preferentially express CSP and hence may contribute to the observed mutant phenotypes. Replacement of dnj-14 with wrmScarlet caused a strong chemotaxis defect, as seen with other dnj-14 null mutants. In contrast, inserting the reporter in-frame to create a DNJ-14-wrmScarlet fusion protein had no effect on chemotaxis, indicating that C-terminal tagging does not impair DNJ-14 function. WrmScarlet fluorescence appeared most obvious in the intestine, head/pharynx, spermathecae and vulva/uterus in the reporter strains, suggesting that DNJ-14 is preferentially expressed in these tissues. Crossing the DNJ-14-wrmScarlet strain with GFP marker strains confirmed the intestinal and pharyngeal expression, but only a partial overlap with neuronal GFP was observed. DNJ-14-wrmScarlet fluorescence in the intestine was increased in response to starvation, which may be relevant to mammalian CSPα's role in microautophagy. DNJ-14's enrichment in worm reproductive tissues (spermathecae and vulva/uterus) parallels the testis-specific expression of CSPβ and CSPγ isoforms in mammals. Furthermore, CSPα messenger RNA is highly expressed in the human proximal digestive tract, suggesting that CSP may have a conserved, but overlooked, function within the gastrointestinal system.

Drosophila Atlastin regulates synaptic vesicle mobilization independent of bone morphogenetic protein signaling

BackgroundThe endoplasmic reticulum (ER) contacts endosomes in all parts of a motor neuron, including the axon and presynaptic terminal, to move structural proteins, proteins that send signals, and lipids over long distances. Atlastin (Atl), a large GTPase, is required for membrane fusion and the structural dynamics of the ER tubules. Atl mutations are the second most common cause of Hereditary Spastic Paraplegia (HSP), which causes spasticity in both sexes’ lower extremities. Through an unknown mechanism, Atl mutations stimulate the BMP (bone morphogenetic protein) pathway in vertebrates and Drosophila. Synaptic defects are caused by atl mutations, which affect the abundance and distribution of synaptic vesicles (SV) in the bouton. We hypothesize that BMP signaling, does not cause Atl-dependent SV abnormalities in Drosophila.ResultsWe show that atl knockdown in motor neurons (Atl-KD) increases synaptic and satellite boutons in the same way that constitutively activating the BMP-receptor Tkv (thick veins) (Tkv-CA) increases the bouton number. The SV proteins Cysteine string protein (CSP) and glutamate vesicular transporter are reduced in Atl-KD and Tkv-CA larvae. Reducing the activity of the BMP receptor Wishful thinking (wit) can rescue both phenotypes. Unlike Tkv-CA larvae, Atl-KD larvae display altered activity-dependent distributions of CSP staining. Furthermore, Atl-KD larvae display an increased FM 1–43 unload than Control and Tkv-CA larvae. As decreasing wit function does not reduce the phenotype, our hypothesis that BMP signaling is not involved is supported. We also found that Rab11/CSP colocalization increased in Atl-KD larvae, which supports the concept that late recycling endosomes regulate SV movements.ConclusionsOur findings reveal that Atl modulates neurotransmitter release in motor neurons via SV distribution independently of BMP signaling, which could explain the observed SV accumulation and synaptic dysfunction. Our data suggest that Atl is involved in membrane traffic as well as formation and/or recycling of the late endosome.

Assessing the Co-Chaperone and Synaptic Protein DNAJC5 as a Therapeutic Target in Alzheimer’s Disease (P1-6.010)

<h3>Objective:</h3> Assess the therapeutic potential of <i>DNAJC5</i> in Alzheimer’s disease (AD). <h3>Background:</h3> <i>DNAJC5</i> encodes cysteine-string protein alpha (CSPα), whose mutations cause adult neuronal ceroid lipofuscinosis (ANCL). ANCL is characterized by endolysosomal dysfunction, synaptic loss, and protein aggregate accumulation, similar to AD. CSPα facilitates secretion of the neurodegenerative proteins tau, TDP-43, and α-synuclein and maintains synaptic integrity. Therefore, CSPα is a promising therapeutic target. <h3>Design/Methods:</h3> N2A695 cells stably expressing mutant <i>DNAJC5</i> or knocking down endogenous <i>Dnajc5</i> were analyzed through immunoblotting and ELISA for APP processing and Aβ production. Brains of ANCL patients and <i>Dnajc5</i>+/−; 5xFAD mice were assessed for amyloid pathology with immunohistochemistry. Transcriptomics from ANCL, AD, and control brains was analyzed for differentially expressed genes (DEGs). The Clue.IO database was used to identify potential targets for gene therapy or pharmacological intervention. <h3>Results:</h3> ANCL-patient brains exhibited low soluble or insoluble Aβ levels, but intra-neuronal Aβ accumulation. They exhibited reductions in synaptic protein levels (SNAP-25, Synaptobrevin, Syntaxin 1, and Synaptophysin). Transcriptomic analysis of ANCL-patient brains showed reduced <i>TSPAN14</i>, which facilitates ADAM10 maturation, elevated <i>APH1B</i>, a γ-secretase subunit, and elevated <i>GSAP</i>, the γ-secretase activating protein. There were 67 upregulated and 15 downregulated transcripts associated with processes essential for synaptic function, including the MAPK cascade (<i>FYN</i>, <i>SOS1</i>), dendritic spine organization (<i>FYN</i>), and endosomal vesicle fusion (<i>VPS11</i>, <i>VPS39</i>). Clue.IO identified heat-shock protein inhibitors and an opioid receptor antagonist (BNTX) as potential pharmacological agents to counteract the effects of mutant <i>DNAJC5</i>. AD patients and 5xFAD mice exhibited reduced <i>DNAJC5</i> expression. Preliminary results from <i>Dnajc5</i>+/−; 5xFAD mice showed increased plaques in the corpus collosum and hippocampus. N2A695 cells expressing <i>DNAJC5</i> mutants showed increased Aβ while <i>DNAJC5</i> knockdown decreased Aβ. <h3>Conclusions:</h3> Low levels of CSPα alter Ab secretion and dysregulate genes and synaptic proteins, suggesting that increasing CSPα levels could reduce Aβ and improve synaptic function. <b>Disclosure:</b> Dr. Rosene has nothing to disclose. Dr. Nykanen has nothing to disclose. Kevin O’Dell has nothing to disclose. Ms. Nunez has nothing to disclose. Dr. Harari has nothing to disclose. John Cirrito has nothing to disclose. Prof. Sands has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for M6P Therapeutics. Prof. Sands has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Amicus. Prof. Sands has stock in JAYA Biosciences. The institution of Prof. Sands has received research support from JAYA Biosciences. Prof. Sands has received intellectual property interests from a discovery or technology relating to health care. Bruno Benitez has nothing to disclose.

Neurotransmitter release progressively desynchronizes in induced human neurons during synapse maturation and aging.

Rapid release of neurotransmitters in synchrony with action potentials is considered a key hardwired property of synapses. Here, in glutamatergic synapses formed between induced human neurons, we show that action potential-dependent neurotransmitter release becomes progressively desynchronized as synapses mature and age. In this solely excitatory network, the emergence of NMDAR-mediated transmission elicits endoplasmic reticulum (ER) stress leading to downregulation of key presynaptic molecules, synaptotagmin-1 and cysteine string protein α, that synchronize neurotransmitter release. The emergence of asynchronous release with neuronal maturity and subsequent aging is maintained by the high-affinity Ca2+ sensor synaptotagmin-7 and suppressed by the introduction of GABAergic transmission into the network, inhibition of NMDARs, and ER stress. These results suggest that long-term disruption of excitation-inhibition balance affects the synchrony of excitatory neurotransmission in human synapses.

Serial Recombineering Cloning to Build Selectable and Tagged Genomic P[acman] BAC Clones for Selection Transgenesis and Functional Gene Analysis using Drosophila melanogaster.

Transgenes with genomic DNA fragments that encompass genes of interest are the gold standard for complementing null alleles in rescue experiments in the fruit fly Drosophila melanogaster. Of particular interest are genomic DNA clones available as bacterial artificial chromosomes (BACs) or fosmids from publicly available genomic DNA libraries. Genes contained within BAC and fosmid clones can be easily modified by recombineering cloning to insert peptide or protein tags to localize, visualize, or manipulate gene products, and to create point mutations or deletions for structure-function analysis of the inserted genes. However, since transgenesis efficiency is inversely correlated with transgene size, obtaining transgenic animals for increasingly larger BAC and fosmid clones requires increasingly laborious screening efforts using the transgenesis marker commonly used for these transgenes, the dominant eye color marker white+ . We recently described a drug-based selectable genetic platform for Drosophila melanogaster, which included four resistance markers that allow direct selection of transgenic animals, eliminating the need to identify transgenic progeny by laborious phenotypic screening. By integrating these resistance markers into BAC transgenes, we were able to isolate animals containing large transgenes by direct selection, avoiding laborious screening. Here we present procedures on how to upgrade BAC clones by serial recombineering cloning to build both selectable and tagged BAC transgenes, for selection transgenesis and functional gene analysis, respectively. We illustrate these procedures using a BAC clone encompassing the gene encoding the synaptic vesicle protein, cysteine string protein. We demonstrate that the modified BAC clone, serially recombineered with a selectable marker for selection transgenesis and an N-terminal green fluorescent protein tag for gene expression analysis, is functional by showing the expression pattern obtained after successful selection transgenesis. The protocols cover: (1) cloning and preparation of the recombineering templates needed for serial recombineering cloning to incorporate selectable markers and protein tags; (2) preparing electrocompetent cells needed to perform serial recombineering cloning; and (3) the serial recombineering workflow to generate both selectable and tagged genomic BAC reporter transgenes for selection transgenesis and functional gene analysis in Drosophila melanogaster. The protocols we describe can be easily adapted to incorporate any of four selectable markers, protein tags, or any other modification for structure-function analysis of the genes present within any of the BAC or fosmid clones. A protocol for generating transgenic animals using serially recombineered BAC clones is presented in an accompanying Current Protocols article (Venken, Matinyan, Gonzalez, & Dierick, 2023a). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning and preparation of recombineering templates used for serial recombineering cloning. Basic Protocol 2: Making electrocompetent cells of the bacterial strains used to perform serial recombineering cloning or induction of plasmid copy number. Basic Protocol 3: Serial recombineering cloning to generate both selectable and tagged genomic P[acman] BAC reporter transgenes for selection transgenesis and gene expression analysis in Drosophila melanogaster.

CSPα in neurodegenerative diseases.

Adult-onset neuronal ceroid lipofuscinosis (ANCL) is a rare neurodegenerative disease characterized by epilepsy, cognitive degeneration, and motor disorders caused by mutations in the DNAJC5 gene. In addition to being associated with ANCL disease, the cysteine string proteins α (CSPα) encoded by the DNAJC5 gene have been implicated in several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease. However, the pathogenic mechanism responsible for these neurodegenerative diseases has not yet been elucidated. Therefore, this study examines the functional properties of the CSPα protein and the related mechanisms of neurodegenerative diseases.

The molecular chaperone cysteine string protein is required for monomeric SNARE proteins to assemble in trans-complexes during human sperm acrosomal exocytosis†.

Membrane fusion in sperm cells is crucial for acrosomal exocytosis and must be preserved to ensure fertilizing capacity. Evolutionarily conserved protein machinery regulates acrosomal exocytosis. Molecular chaperones play a vital role in spermatogenesis and post-testicular maturation. Cysteine string protein (CSP) is a member of the Hsp40 co-chaperones, and the participation of molecular chaperones in acrosomal exocytosis is poorly understood. In particular, the role of CSP in acrosomal exocytosis has not been reported so far. Using western blot and indirect immunofluorescence, we show that CSP is present in human sperm, is palmitoylated, and predominantly bound to membranes. Moreover, using functional assays and transmission electron microscopy, we report that blocking the function of CSP avoided the assembly of trans-complexes and inhibited exocytosis. In summary, here, we describe the presence of CSP in human sperm and show that this protein has an essential role in membrane fusion during acrosomal exocytosis mediating the trans-SNARE complex assembly between the outer acrosomal and plasma membranes. In general, understanding CSP's role is critical in identifying new biomarkers and generating new rational-based approaches to treat male infertility.

A Caenorhabditis elegans model of autosomal dominant adult-onset neuronal ceroid lipofuscinosis identifies ethosuximide as a potential therapeutic.

Autosomal dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is a rare neurodegenerative disorder characterized by progressive dementia and premature death. Four ANCL-causing mutations have been identified, all mapping to the DNAJC5 gene that encodes cysteine string protein α (CSPα). Here, using Caenorhabditis elegans, we describe an animal model of ANCL in which disease-causing mutations are introduced into their endogenous chromosomal locus, thereby mirroring the human genetic disorder. This was achieved through CRISPR/Cas9-mediated gene editing of dnj-14, the C. elegans ortholog of DNAJC5. The resultant homozygous ANCL mutant worms exhibited reduced lifespans and severely impaired chemotaxis, similar to isogenic dnj-14 null mutants. Importantly, these phenotypes were also seen in balanced heterozygotes carrying one wild-type and one ANCL mutant dnj-14 allele, mimicking the heterozygosity of ANCL patients. We observed a more severe chemotaxis phenotype in heterozygous ANCL mutant worms compared with haploinsufficient worms lacking one copy of CSP, consistent with a dominant-negative mechanism of action. Additionally, we provide evidence of CSP haploinsufficiency in longevity, as heterozygous null mutants exhibited significantly shorter lifespan than wild-type controls. The chemotaxis phenotype of dnj-14 null mutants was fully rescued by transgenic human CSPα, confirming the translational relevance of the worm model. Finally, a focused compound screen revealed that the anti-epileptic drug ethosuximide could restore chemotaxis in dnj-14 ANCL mutants to wild-type levels. This suggests that ethosuximide may have therapeutic potential for ANCL and demonstrates the utility of this C. elegans model for future larger-scale drug screening.

The CD63 homologs, Tsp42Ee and Tsp42Eg, restrict endocytosis and promote neurotransmission through differential regulation of synaptic vesicle pools.

The Tetraspanin (Tsp), CD63, is a transmembrane component of late endosomes and facilitates vesicular trafficking through endosomal pathways. Despite being widely expressed in the human brain and localized to late endosomes, CD63's role in regulating endo- and exocytic cycling at the synapse has not been investigated. Synaptic vesicle pools are highly dynamic and disruptions in the mobilization and replenishment of these vesicle pools have adverse neuronal effects. We find that the CD63 homologs, Tsp42Ee and Tsp42Eg, are expressed at the Drosophila neuromuscular junction to regulate synaptic vesicle pools through both shared and unique mechanisms. Tsp42Ee and Tsp42Eg negatively regulate endocytosis and positively regulate neurotransmitter release. Both tsp mutants show impaired locomotion, reduced miniature endplate junctional current frequencies, and increased endocytosis. Expression of human CD63 in Drosophila neurons leads to impaired endocytosis suggesting the role of Tsps in endocytosis is conserved. We further show that Tsps influence the synaptic cytoskeleton and membrane composition by regulating Futsch loop formation and synaptic levels of SCAR and PI(4,5)P2. Finally, Tsp42Ee and Tsp42Eg influence the synaptic localization of several vesicle-associated proteins including Synapsin, Synaptotagmin, and Cysteine String Protein. Together, our results present a novel function for Tsps in the regulation of vesicle pools and provide insight into the molecular mechanisms of Tsp-related synaptic dysfunction.

Activity-induced synaptic structural modifications by Akt

The activity-dependent regulation of synaptic structures plays a key role in synaptic development and plasticity; however, the signaling mechanisms involved remain largely unknown. The serine/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K), plays a pivotal role in a wide range of physiological functions. We focused on the importance of Akt in rapid synaptic structural changes after stimulation at the Drosophila neuromuscular junction, a well-studied model synapse. Compared with wild-type larvae, akt mutants showed significantly reduced muscle size and an increased number of boutons per area, suggesting that Akt is required for proper pre- and postsynaptic growth. In addition, the level of cysteine string protein (CSP) was significantly increased, and its distribution was different in akt mutants. After high K+ single stimulation, the CSP level of akt mutant NMJs increased dramatically compared with that of wild-type NMJs. Interestingly, ghost boutons without postsynaptic specialization were found in akt mutant NMJs, and the number of these boutons was significantly increased by patterned stimulation. In contrast, the postsynaptic change in the subsynaptic reticulum (SSR) in the akt mutant occurred independent of stimulation. These results suggest that Akt functions in both pre- and postsynaptic growth and differentiation, and in particular, presynaptic action occurs in an activity-dependent manner.

Cysteine string protein alpha accumulates with early pre-synaptic dysfunction in Alzheimer's disease.

In Alzheimer’s disease, synapse loss causes memory and cognitive impairment. However, the mechanisms underlying synaptic degeneration in Alzheimer’s disease are not well understood. In the hippocampus, alterations in the level of cysteine string protein alpha, a molecular co-chaperone at the pre-synaptic terminal, occur prior to reductions in synaptophysin, suggesting that it is a very sensitive marker of synapse degeneration in Alzheimer’s. Here, we identify putative extracellular accumulations of cysteine string alpha protein, which are proximal to beta-amyloid deposits in post-mortem human Alzheimer’s brain and in the brain of a transgenic mouse model of Alzheimer’s disease. Cysteine string protein alpha, at least some of which is phosphorylated at serine 10, accumulates near the core of beta-amyloid deposits and does not co-localize with hyperphosphorylated tau, dystrophic neurites or glial cells. Using super-resolution microscopy and array tomography, cysteine string protein alpha was found to accumulate to a greater extent than other pre-synaptic proteins and at a comparatively great distance from the plaque core. This indicates that cysteine string protein alpha is most sensitive to being released from pre-synapses at low concentrations of beta-amyloid oligomers. Cysteine string protein alpha accumulations were also evident in other neurodegenerative diseases, including some fronto-temporal lobar dementias and Lewy body diseases, but only in the presence of amyloid plaques. Our findings are consistent with suggestions that pre-synapses are affected early in Alzheimer’s disease, and they demonstrate that cysteine string protein alpha is a more sensitive marker for early pre-synaptic dysfunction than traditional synaptic markers. We suggest that cysteine string protein alpha should be used as a pathological marker for early synaptic disruption caused by beta-amyloid.

Cysteine String Protein Controls Two Routes of Export for Misfolded Huntingtin.

Extracellular vesicles (EVs) are secreted vesicles of diverse size and cargo that are implicated in the cell-to-cell transmission of disease-causing-proteins in several neurodegenerative diseases. Mutant huntingtin, the disease-causing entity in Huntington’s disease, has an expanded polyglutamine track at the N terminus that causes the protein to misfold and form toxic intracellular aggregates. In Huntington’s disease, mutant huntingtin aggregates are transferred between cells by several routes. We have previously identified a cellular pathway that is responsible for the export of mutant huntingtin via extracellular vesicles. Identifying the EV sub-populations that carry misfolded huntingtin cargo is critical to understanding disease progression. In this work we expressed a form of polyglutamine expanded huntingtin (GFP-tagged 72Qhuntingtinexon1) in cells to assess the EVs involved in cellular export. We demonstrate that the molecular chaperone, cysteine string protein (CSPα; DnaJC5), facilitates export of disease-causing-polyglutamine-expanded huntingtin cargo in 180–240 nm vesicles as well as larger 10–30 μm vesicles.

CSPα reduces aggregates and rescues striatal dopamine release in α-synuclein transgenic mice.

α-Synuclein aggregation at the synapse is an early event in Parkinson’s disease and is associated with impaired striatal synaptic function and dopaminergic neuronal death. The cysteine string protein (CSPα) and α-synuclein have partially overlapping roles in maintaining synaptic function and mutations in each cause neurodegenerative diseases. CSPα is a member of the DNAJ/HSP40 family of co-chaperones and like α-synuclein, chaperones the SNARE complex assembly and controls neurotransmitter release. α-Synuclein can rescue neurodegeneration in CSPαKO mice. However, whether α-synuclein aggregation alters CSPα expression and function is unknown. Here we show that α-synuclein aggregation at the synapse is associated with a decrease in synaptic CSPα and a reduction in the complexes that CSPα forms with HSC70 and STGa. We further show that viral delivery of CSPα rescues in vitro the impaired vesicle recycling in PC12 cells with α-synuclein aggregates and in vivo reduces synaptic α-synuclein aggregates increasing monomeric α-synuclein and restoring normal dopamine release in 1-120hαSyn mice. These novel findings reveal a mechanism by which α-synuclein aggregation alters CSPα at the synapse, and show that CSPα rescues α-synuclein aggregation-related phenotype in 1-120hαSyn mice similar to the effect of α-synuclein in CSPαKO mice. These results implicate CSPα as a potential therapeutic target for the treatment of early-stage Parkinson’s disease.

Presynaptic accumulation of α-synuclein causes synaptopathy and progressive neurodegeneration in Drosophila.

Alpha-synuclein (α-syn) mislocalization and accumulation in intracellular inclusions is the major pathological hallmark of degenerative synucleinopathies, including Parkinson’s disease, Parkinson’s disease with dementia and dementia with Lewy bodies. Typical symptoms are behavioural abnormalities including motor deficits that mark disease progression, while non-motor symptoms and synaptic deficits are already apparent during the early stages of disease. Synucleinopathies have therefore been considered synaptopathies that exhibit synaptic dysfunction prior to neurodegeneration. However, the mechanisms and events underlying synaptopathy are largely unknown. Here we investigated the cascade of pathological events underlying α-syn accumulation and toxicity in a Drosophila model of synucleinopathy by employing a combination of histological, biochemical, behavioural and electrophysiological assays. Our findings demonstrate that targeted expression of human α-syn leads to its accumulation in presynaptic terminals that caused downregulation of synaptic proteins, cysteine string protein, synapsin, and syntaxin 1A, and a reduction in the number of Bruchpilot puncta, the core component of the presynaptic active zone essential for its structural integrity and function. These α-syn-mediated presynaptic alterations resulted in impaired neuronal function, which triggered behavioural deficits in ageing Drosophila that occurred prior to progressive degeneration of dopaminergic neurons. Comparable alterations in presynaptic active zone protein were found in patient brain samples of dementia with Lewy bodies. Together, these findings demonstrate that presynaptic accumulation of α-syn impairs the active zone and neuronal function, which together cause synaptopathy that results in behavioural deficits and the progressive loss of dopaminergic neurons. This sequence of events resembles the cytological and behavioural phenotypes that characterise the onset and progression of synucleinopathies, suggesting that α-syn-mediated synaptopathy is an initiating cause of age-related neurodegeneration.

Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone Hsc70-5 in Drosophila

ABSTRACT We investigated in larval and adult Drosophila models whether loss of the mitochondrial chaperone Hsc70-5 is sufficient to cause pathological alterations commonly observed in Parkinson disease. At affected larval neuromuscular junctions, no effects on terminal size, bouton size or number, synapse size, or number were observed, suggesting that we studied an early stage of pathogenesis. At this stage, we noted a loss of synaptic vesicle proteins and active zone components, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements. The adult model displayed ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. Adult phenotypes could be suppressed by knockdown of dj-1β, Lrrk, DCTN2-p50, DCTN1-p150, Atg1, Atg101, Atg5, Atg7, and Atg12. The knockdown of components of the macroautophagy/autophagy machinery or overexpression of human HSPA9 broadly rescued larval and adult phenotypes, while disease-associated HSPA9 variants did not. Overexpression of Pink1 or promotion of autophagy exacerbated defects. Abbreviations: AEL: after egg laying; AZ: active zone; brp: bruchpilot; Csp: cysteine string protein; dlg: discs large; eEJPs: evoked excitatory junctional potentials; GluR: glutamate receptor; H2O2: hydrogen peroxide; mEJP: miniature excitatory junctional potentials; MT: microtubule; NMJ: neuromuscular junction; PD: Parkinson disease; Pink1: PTEN-induced putative kinase 1; PSD: postsynaptic density; SSR: subsynaptic reticulum; SV: synaptic vesicle; VGlut: vesicular glutamate transporter.

α‐Synuclein’s Role in Platelet Exocytosis

Platelets use SNARE‐mediated exocytosis to maintain hemostasis and thrombosis. These processes are maintained by the exocytosis of platelet releasate from the three types of granules in platelets: dense, alpha, and lysosomal. Understanding how the process of exocytosis is regulated in secretion, we look for potential SNARE regulators and found the protein α‐synuclein. α‐Synuclein appears to be the only member of the synuclein family present in platelets and is very abundant. To address the role of α‐synuclein in platelets, we examined the hemostatic phenotype of α‐synuclein−/− mice. Using a tail‐bleeding time assay, we observed that α‐synuclein−/− mice bleed for a slightly longer time compared to the wild‐type mice, suggesting a platelet granule secretion defect. Secretion kinetic and dose‐response assays showed that platelets from α‐synuclein−/− mice have defective release from the dense granules and less so from lysosomal granules. To understand the mechanism of this defect, we asked whether α‐synuclein is acting as a VAMP‐chaperone in platelets and thus modulates secretion by controlling V‐SNARE levels. The two dominant T‐SNARES SNAP‐23 and Syntaxin‐11 are not altered in α‐synuclein−/− mice. However, the dominant V‐SNARE VAMP‐8 was reduced. Other V‐SNARES in the α‐synuclein−/− mice VAMP‐2, VAMP‐3, and VAMP‐7 were normal. Additional western blotting experiments demonstrated the presence of the α‐synuclein‐interactor Cysteine String Protein, further identifying elements of the platelet secretory machinery. These experiments demonstrate a role for α‐synuclein in platelet exocytosis and hemostasis and will further fill a gap in our knowledge on α‐synuclein’s physiological function and understanding how the process of exocytosis is regulated.Support or Funding InformationThis work was supported by a grant from the National Institutes of Health, National Heart, Lung, and Blood Institute (HL56652) and a Veterans Affairs Merit Award to S.W.W.

Cysteine string proteins.

Cysteine string protein (CSP) was discovered by use of a synapse-specific, monoclonal antibody to screen a cDNA expression library in Drosophila. A vertebrate CSP homolog was later identified and shown to co-purify with synaptic vesicles. CSP-α is now recognized as a membrane constituent of many regulated secretory organelles. Knockout of the csp gene in Drosophila produced temperature-sensitive paralysis reflecting a loss of evoked (but not spontaneous) transmitter release. However, CSP's role in regulated exocytosis remains ambiguous. Fruit flies lacking the csp gene also exhibited nerve terminal degeneration as did mice deficient in the csp-α gene. This phenotype has been ascribed to the depletion of a functional pool of the t-SNARE, SNAP-25. However, recent studies showing that an endosomal pool of CSP-α contributes to a novel, protein-export pathway argues that CSP's role in neurodegeneration is more complex. Clients of this later pathway include tau and α-synuclein, proteins linked to neurodegeneration. Additionally, mutations in the csp-α gene cause an adult-onset, neuronal ceroid lipofuscinosis and diminished CSP-α expression is an early event in Alzheimer's disease. Collectively, these findings indicate that much remains to be learned about the role of CSPs in cellular secretory pathways and human disease.

Autosomal-dominant adult neuronal ceroid lipofuscinosis caused by duplication in DNAJC5 initially missed by Sanger and whole-exome sequencing.

Adult-onset neuronal ceroid lipofuscinoses (ANCL, Kufs disease) are rare hereditary neuropsychiatric disorders characterized by intralysosomal accumulation of ceroid in tissues. The ceroid accumulation primarily affects the brain, leading to neuronal loss and progressive neurodegeneration. Although several causative genes have been identified (DNAJC5, CLN6, CTSF, GRN, CLN1, CLN5, ATP13A2), the genetic underpinnings of ANCL in some families remain unknown. Here we report one family with autosomal dominant (AD) Kufs disease caused by a 30 bp in-frame duplication in DNAJC5, encoding the cysteine-string protein alpha (CSPα). This variant leads to a duplication of the central core motif of the cysteine-string domain of CSPα and affects palmitoylation-dependent CSPα sorting in cultured neuronal cells similarly to two previously described CSPα variants, p.(Leu115Arg) and p.(Leu116del). Interestingly, the duplication was not detected initially by standard Sanger sequencing due to a preferential PCR amplification of the shorter wild-type allele and allelic dropout of the mutated DNAJC5 allele. It was also missed by subsequent whole-exome sequencing (WES). Its identification was facilitated by reanalysis of original WES data and modification of the PCR and Sanger sequencing protocols. Independently occurring variants in the genomic sequence of DNAJC5 encoding the cysteine-string domain of CSPα suggest that this region may be more prone to DNA replication errors and that insertions or duplications within this domain should be considered in unsolved ANCL cases.